Tuberculosis 2007

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Palomino – Leão – Ritacco Tuberculosis 2007 From basic science to patient care TuberculosisTextbook.com First Edition

This textbook was made possible by an unrestricted educational grant provided by Bernd Sebastian Kamps and Patricia Bourcillier.

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Tuberculosis 2007

From basic science to patient care

www.TuberculosisTextbook.com

Juan Carlos Palomino

Sylvia Cardoso Leão

Viviana Ritacco

(Editors)

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Editors

Juan Carlos Palomino

Mycobacteriology Unit Institute of Tropical Medicine Nationalestraat, 155

20000, Antwerp BELGIUM

Sylvia Cardoso Leão

Departamento de Microbiologia, Imunologia e Parasitologia Universidade Federal de São Paulo

Rua Botucatu, 862 3° andar 04023-062, São Paulo, SP BRAZIL

Viviana Ritacco

Servicio de Micobacterias, Instituto Nacional de Enfermedades Infecciosas Carlos G. Malbrán

Av. Velez Sarsfield, 563 1281, Buenos Aires ARGENTINA

Cover design by Pedro Cardoso Leão, BRAZIL (pedrocardosoleao@yahoo.com.br)

Disclaimer

Tuberculosis is an ever-changing field. The editors and authors of “Tuberculosis 2007 – from basic science to patient care” have made every effort to provide information that is accurate and complete as of the date of publication. However, in view of the rapid changes occurring in medical science, as well as the possibility of human error, this site may contain technical inaccuracies, typographical or other errors. Readers are advised to check the product informa-tion currently provided by the manufacturer of each drug to be administered to verify the recommended dose, the method and duration of administration, and contraindications. It is the responsibility of the treating physician who relies on experience and knowledge about the patient to determine dosages and the best treatment for the patient. The information con-tained herein is provided "as is" and without warranty of any kind. The contributors to this site disclaim responsibility for any errors or omissions or for results obtained from the use of in-formation contained herein.

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Preface

This book is the result of a joint effort in response to the Amedeo Challenge to write and publish a medical textbook on tuberculosis. This non-profit-making ini-tiative is particularly attractive due to several reasons. First, the medium chosen for dissemination: the book will be readily available on the internet and access will be free to anyone. Second, its advantage over books published via traditional media is the ease to update the information on a regular basis. Third, with the exception of Spanish and Portuguese, no copyright is allocated and the translation of Tuberculo-sis 2007 to all other languages is highly encouraged.

These innovations in the way of publication were translated to the organization of the chapters in the book. This is not a classical textbook on tuberculosis diagno-sis, management, and treatment. On the contrary, it is a multidisciplinary approach addressing a full range of topics, from basic science to patient care. Most authors are former members of RELACTB – a Tuberculosis Research Network for Latin America, the Caribbean and Europe sponsored by the United Nations University − and have worked on collaborative projects since 1995.

Classical knowledge about the disease is focused on chapters dedicated to the history of tuberculosis, microbiology of the tubercle bacillus, description of the disease caused by Mycobacterium tuberculosis complex members in adults, chil-dren, and HIV/AIDS patients, conventional epidemiology, diagnostics, biosafety, and treatment.

More recent findings, which have changed our knowledge about tuberculosis in the last years, are detailed in chapters on the molecular evolution of the M. tuber-culosis complex, molecular epidemiology, host genetics, immune response and susceptibility to tuberculosis, studies on the pathogenesis of tuberculosis in animal models, and new diagnostic and drug resistance detection approaches.

Perspectives for future research relevant to fighting the disease have also been included in chapters focusing on the “omics” technologies, from genomics to pro-teomics, metabolomics and lipidomics, and on research dedicated to the develop-ment of new vaccines and new diagnostic methods, and are discussed in the last chapter.

Nowadays, medical science should not be limited to academic circles but read-ily translated into practical applications aimed at patient care and control of dis-ease. Thus, we expect that our initiative will stimulate the interest of readers not only in solving clinical topics on the management of tuberculosis but also in posing new questions back to basic science, fostering a continuous bi-directional interac-tion of medical care, and clinical and basic research.

Juan Carlos Palomino, Sylvia Cardoso Leão, Viviana Ritacco Belgium, Brazil, Argentina – June 2007

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Contributing Authors

Afrânio Kritski, MD, PhD

Unidade de Pesquisas em Tuberculose, Hospital Clementino Fraga Filho, Departamento de Clinica Medica, Faculdade de Medicina, Universidade Federal do Rio de Janeiro (UFRJ), Av Professor Rodolpho Rocco s/n – Ilha do Fundão 4º andar, 21941-590, Rio de Janeiro, BRAZIL

Phone: ++ 55 21 2562 2426 – Fax: ++ 55 21 25506903 kritskia@gmail.com

Angel Adrián Cataldi, PhD

Centro de Investigacciones en Ciencias Veterinarias y Agrarias (CICVyA), Instituto Nacional de Tecnologia Agropecuaria (INTA), Los Reseros y Las Cabañas (1712) Castelar, ARGENTINA

Phone: ++ 54 11 4621 0199 – Fax: ++ 54 11 4621 0199 acataldi@cnia.inta.gov.ar

Alejandro Reyes, MSc Biology

Grupo de Biotecnología Molecular, Corporación Corpogen, Carrera 5 No 66A-34, Bogotá D.C., COLOMBIA

Phone: ++ 57 1 3484610 – Fax: ++ 57 1 3484607 alejandroreyesmunoz@gmail.com

Anandi Martin, PhD

Mycobacteriology Unit, Institute of Tropical Medicine, Nationalestraat, 155, 2000, Antwerp, BELGIUM

Phone: ++ 32 3 2476334 – Fax: ++ 32 3 247 6333 amartin@itg.be

Brigitte Gicquel, PhD

Unité de Génétique Mycobactérienne, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris-Cedex 15, FRANCE

Phone: ++ 33 1 45688828 – Fax: ++ 33 1 45688843 bgicquel@pasteur.fr

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Carlos Martin, MD, PhD

Grupo de Genetica de Micobacterias, Departamento de Microbiologia, Medicina Preventiva y Salud Publica, Facultad de Medicina, Universidad de Zaragoza, C/ Domingo Miral sn, 50009, Zaragoza, SPAIN

Phone: ++ 34 976 761759 – Fax: ++ 34 976 761664 carlos@unizar.es

Christophe Sola, PharmD, PhD*

Unité de la Tuberculose & des Mycobactéries, Institut Pasteur de Guade-loupe, Morne Joliviere, BP 484, 97183-Abymes, Cedex, GUADELOUPE * Current affiliation : Unité de Génétique Mycobactérienne, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris-Cedex 15, FRANCE

Phone: ++ 33 1 40613274 – Fax: ++ 33 1 45688843 csola@pasteur.fr

Clara Inés Espitia, PhD

Instituto de Investigaciones Biomédicas, Apartado Postal 70228, Ciudad Universitaria 04510, Mexico D.F., MEXICO

Phone: ++ 52 55 6223818 – Fax: ++ 52 55 6223369 espitia@servidor.unam.mx

Dick van Soolingen, PhD

Mycobacteria Reference Unit, Centre for Infectious Disease Control (CIb/LIS), National Institute of Public Health and the Environment (RIVM), P.O. box 1, 3720 BA Bilthoven, THE NETHERLANDS

Phone: ++ 31 30 2742363 – Fax: ++ 31 30 2744418 D.van.Soolingen@rivm.nl

Domingo Palmero, MD, PhD

Hospital de Enfermedades Infecciosas F. J. Muñiz, Uspallata 2272 (C1282AEN), Buenos Aires, ARGENTINA.

Phone: ++ 54 11 44326569 – Fax: ++ 54 11 44326569 djpalmero@intramed.net

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Contributing Authors 9

Enrico Tortoli, ScD

Regional Reference Center for Mycobacteria. Careggi Hospital. Viale Mor-gagni 85, 50134 Florence, ITALY

Phone: ++ 39 055 7949199 – Fax: ++ 39 055 7949010 e.tortoli@libero.it

Ernesto Montoro, MD, PhD

Instituto de Medicina Tropical ¨Pedro Kourí¨. Autopista Novia del Mediodia Km 6 ½, La Lisa, Ciudad de La Habana, CUBA

Phone: ++ 53 7 2020651 – Fax: ++ 53 7 2046051 emontoro@ipk.sld.cu

Fabiana Bigi, PhD

Phone: ++ 54 11 46211447 – Fax: ++ 54 11 46210199 fbigi@cnia.inta.gov.ar

Fernando Augusto Fiuza de Melo, MD, PhD

Instituto Clemente Ferreira, Rua da Consolação, 717, 01301-000, São Paulo, BRAZIL

Phone: ++ 55 11 32190750 – Fax: ++ 55 11 38857827 fernandofiuza@terra.com.br

Françoise Portaels, PhD

Phone: ++ 32 3 2476317 – Fax: ++ 32 3 247 6333 portaels@itg.be

Howard E. Takiff, MD, MPH

Centro de Microbiología y Biología Celular (CMBC), Instituto Venezolano de Investigaciones Científicas (IVIC), Km. 11 Carr. Panamericana, Cara-cas, 1020A, VENEZUELA.

Phone: ++ 58 212 5041439 – Fax: ++ 58 212 5041382 htakiff@ivic.ve

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Iris Estrada-García, PhD

Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional. Prol. de Carpio y Plan de Ayala S/N, Mexico DF, MEXICO C.P. 11340

Phone: ++ 52 55 57296300 ext. 62507 – Fax: ++ 52 55 57296300, ext. 46211

iestrada@encb.ipn.mx and iestrada5@hotmail.com

Jacobus H. de Waard, PhD

Laboratorio de Tuberculosis, Instituto de Biomedicina, Al lado de Hospital Vargas, San José, Caracas, VENEZUELA

Phone: ++ 58 212 8306670 – Fax: ++ 58 212 8611258 jacobusdeward@movistar.net.ve

Jaime Robledo, MD

Unidad de Bacteriología y Micobacterias, Corporación para Investigaciones Biológicas, Carrera 72A No.78B-141, Medellín, COLOMBIA.

Phone: ++ 57 4 4410855 ext. 216 – Fax: ++ 57 4 4415514 jrobledo@cib.org.co

Jeanet Serafín-López, PhD

Phone: ++ 52 55 57296300, ext. 62369 – Fax: ++ 52 55 57296300 ext. 46211

jeaserafin@hotmail.com

José-Antonio Aínsa Claver, PhD

Grupo de Genética de Micobacterias, Departamento de Microbiología, Medicina Preventiva y Salud Pública, Facultad de Medicina, Universidad de Zaragoza. C/Domingo Miral s/n, 50009, Zaragoza, SPAIN.

Phone: ++ 34 976 761742 – Fax: ++ 34 976 761604 ainsa@unizar.es

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Juan Carlos Palomino, PhD

Phone: ++32 3 2476334 – Fax: ++ 32 3 247 6333 palomino@itg.be

Kristin Kremer, PhD

Mycobacteria Reference Unit, Centre for Infectious Disease Control (CIb/LIS), National Institute of Public Health and the Environment (RIVM), P.O. box 1, 3720 BA Bilthoven, THE NETHERLANDS

Phone: ++ 31 30 2742720 – Fax: ++ 31 30 2744418 Kristin.Kremer@rivm.nl

Leiria Salazar, PhD

Departamento de Biología Estructural, Instituto Venezolano de Investiga-ciones Científicas (IVIC), Apartado 21827, Caracas, 1020A, VENEZUELA Phone: ++ 58 212 5041715 – Fax: ++ 58 212 5041444

lsalazar@ivic.ve

Lucía Elena Barrera, Lic Biol

Servicio de Micobacterias, Instituto Nacional de Enfermedades Infecciosas Carlos G. Malbrán, Av. Velez Sarsfield 563 (1281) Buenos Aires, ARGEN-TINA

Phone: ++ 54 11 43027635 – Fax: ++ 54 11 43027635 lbarrera@anlis.gov.ar

Mahavir Singh, PhD

Department of Genome Analysis, Helmholtz Center for Infection Research (former GBF), and LIONEX GmbH, Inhoffenstr. 7, 38124 Braunschweig, GERMANY

Phone : ++ 49 531 61815320 – Fax : ++ 49 531 2601159 msi@helmholtz-hzi.de and info@lionex.de

Maria Alice da Silva Telles, Lic Biol

Setor de Micobactérias, Instituto Adolfo Lutz, Av. Dr. Arnaldo, 355, 01246-902, São Paulo SP, BRAZIL

Phone: ++ 55 11 30682895 – Fax: ++ 55 11 30682892 atelles@ial.sp.gov.br

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María del Carmen Menéndez, MSc, PhD

Departamento de Medicina Preventiva. Facultad de Medicina, Universidad Autonoma de Madrid, Arzobispo Morcillo, 4, 28029-Madrid, SPAIN

Phone: ++ 34 914 975491 – Fax: ++ 34 914 975353 carmen.menendez@uam.es

María Isabel Romano, PhD

Phone: ++ 54 11 46211447 – Fax: ++ 54 11 46210199 mromano@cnia.inta.gov.ar

María Jesús García, MD, MSc, PhD

Departamento de Medicina Preventiva. Facultad de Medicina, Universidad Autonoma de Madrid, Arzobispo Morcillo, 4, 28029-Madrid, SPAIN

Phone: ++ 34 914 975491 – Fax: ++ 34 914 975353 mariaj.garcia@uam.es

Nalin Rastogi, MSc, PhD, DSc

Unité de la Tuberculose & des Mycobactéries, Institut Pasteur de Guade-loupe, Morne Joliviere, BP 484, 97183-Abymes, Cedex, GUADELOUPE Phone: ++ 590 590 893881 – Fax: ++ 590 590 893880

nrastogi@pasteur-guadeloupe.fr

Nora Morcillo, PhD

Reference Laboratory of Tuberculosis Control Program of Buenos Aires Province, Hospital Dr. Cetrangolo, Vicente Lopez (1602) Buenos Aires, ARGENTINA

Phone: ++ 54 11 4970165 – Fax: ++ 54 11 47219153 nora_morcillo@yahoo.com.ar

Patricia Del Portillo Obando, Lic Microbiol

Corporación Corpogen, Carrera 5 No 66A-34, Bogota, D.C. COLOMBIA Phone: ++ 57 1 3484609/06 – Fax: ++ 57 1 3484607

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Pedro Eduardo Almeida da Silva, PhD

Laboratório de Micobactérias, Departamento de Patologia, Fundação Uni-versidade Federal do Rio Grande (FURG), Rua General Osório S/N, Cam-pus Saúde, Rio Grande RS, BRAZIL

Phone: ++ 55 53 32338895 – Fax: ++ 55 53 32338860 pedre@furg.br and pedre_99@yahoo.com.br

Peter WM Hermans, PhD

Laboratory of Pediatric Infectious Diseases, Radboud University Nijmegen Medical Centre, PO Box 9101 (internal post 224), 6500 HB Nijmegen, THE NETHERLANDS

Phone: ++ 31 24 3666406 (office), ++ 31 24 3666407 (secr.) Fax: ++ 31 24 3666352

P.Hermans@cukz.umcn.nl

Rodolfo Rodríguez Cruz, MD

Organização Pan-Americana da Saude, Setor de Embaixadas Norte, Lote 19, 70800-400, Brasília DF, BRAZIL

Phone: ++ 55 61 34269546 – Fax: ++ 55 61 34269591 rodolfor@bra.ops-oms.org

Rogelio Hernández-Pando, MD, PhD

Seccion de Patología Experimental, Departamento de Patologia, Instituto Nacional de Ciencias Médicas y Nutricion Salvador Zubiran, Vasco de Qui-roga no 15, Tlalpan, CP-14000, Mexico DF, MEXICO.

Phone: ++ 52 55 54853491 – Fax: ++ 52 55 56551076 rhdezpando@hotmail.com and rhpando@quetzal.innsz.mx

Rommel Chacon-Salinas, PhD

Phone: ++ 52 55 57296300, ext. 62369 – Fax: ++ 52 55 57296300, ext. 46211

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Sylvia Cardoso Leão, MD, MSc, PhD

Departamento de Microbioloiga, Imunologia e Parasitologia, Universidade Federal de São Paulo (UNIFESP), Rua Botucatu 862, 3° andar, 04023-062, São Paulo SP, BRAZIL

Phone: ++ 55 11 55764537 – Fax: ++ 55 11 55724711 sylvia@ecb.epm.br

Viviana Ritacco, MD, PhD

Servicio de Micobacterias, Instituto Nacional de Enfermedades Infecciosas Car-los G. Malbrán, Av. Velez Sarsfield 563 (1281) Buenos Aires, ARGENTINA Phone: ++ 54 11 43027635 – Fax: ++ 54 11 43027635

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Abbreviations

2-DE: two dimensional electrophoresis ADA: adenosine deaminase

ADC: albumin, dextrose, catalase AFB: acid fast bacilli

AIDS: acquired immunodeficiency syndrome

AMTD: Amplified Mycobacterium tuberculosis Direct Test BAC: bacterial artificial chromosome

BCG: bacille Calmette-Guérin bp: base pair

cAMP: cyclic adenosine monophosphate CAS: Central-Asian (or Delhi)

CD4+: cluster of differentiation 4 glicoprotein CD8+: cluster of differentiation 8 glicoprotein CDC: Centers for Disease Control and Prevention cfu: colony forming units

CMI: cell mediated immunity CPC: cetylpyridinium chloride CPF-10: culture filtrate protein 10 CR: complement receptor

CRISPR: clustered regularly interspersed palindromic repeats CTL: cytotoxic T lymphocyte

DARQ: diarylquinoline DAT: diacyl trehalose

DC-SIGN: dendritic cell-specific intercellular-adhesion-molecule-grabbing non-integrin DNA: desoxyribonucleic acid

DOTS: directly observed therapy short-course DR: direct repeat

DST: drug susceptibility test DTH: delayed type hypersensitivity EAI: East-African-Indian

EDTA: ethylenediaminetetraacetic acid ELISA: enzyme-linked immunosorbent assay

ELISPOT: enzyme-linked immunospot for interferon-gamma EMB: ethambutol

ESAT-6: 6 kDa early secretory antigenic target ETH: ethionamide

Fc: crystallizable fraction of the Ig molecule FDA: Food and Drug Administration FGF-2: fibroblast growth factor 2

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G+C: guanine plus cytosine GLC: gas-liquid chromatography GLP: good laboratory practices

HAART: highly active anti-retroviral therapy HEPA: high efficiency particulate air HIV: human immunodeficiency virus HLA: human leukocyte antigen

HPLC: high-performance liquid chromatography Hsp: heat-shock protein

IATA: International Air Transportation Association ICAT: isotope-coded affinity tag

IFN-γ: interferon-gamma

IFN-γR: interferon-gamma receptor InhA: enoyl acyl carrier protein reductase Ig: immunoglobulin

IL: interleukin INH: isoniazid

iNOS: inducible nitric oxide synthase

IS6110 RFLP: restriction fragment length polymorphism based on insertion sequence IS6110 ITS: internal transcribed spacer

IUATLD: International Union Against Tuberculosis and Lung Disease KasA: beta-ketoacyl ACP synthase

KatG: catalase-peroxidase enzyme kDa: kiloDalton

LC: liquid chromatography LSP: large sequence polymorphism mAG: mycolyl-arabinogalactan

MALDI: matrix assisted laser desorption/ionization MBL: mannose-binding lectin

MCP-1: monocyte chemoattractant protein-1 MDR: multidrug-resistant

MGIT: Mycobacteria Growth Indicator Tube MHC: major histocompatibility complex MIC: minimal inhibitory concentration

MIRU: mycobacterial interspersed repetitive units MLST: multilocus sequence typing

MODS: microscopic observation broth-drug susceptibility assay mRNA: messenger ribonucleic acid

MS: mass spectrometry

MSMD: mendelian susceptibility to mycobacterial diseases MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

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Abbreviations 17

NEMO: NF-κB essential modulator NF-κ-B: nuclear factor kappa B NO: nitric oxide

NRP-1: microaerobic stage of nonreplicating persistence NRP-2: anaerobic state

NRAMP1: natural resistance-associated macrophage protein 1 nsSNP: non-synonymous single nucleotide polymorphism NTM: non-tuberculous mycobacteria

OADC: oleic acid, albumin, dextrose, catalase OD: optical density

ORF: open reading frame

oriC: origin of replication

PANTA: polymyxin B, amphotericin B, nalidixic acid, trimethoprim, azlocillin PAS: para-aminosalycilic acid

PAT: penta-acyl trehalose PCR: polymerase chain reaction PDIM: phthiocerol dimycocerosate PE: proteins that have the motif Pro-Glu PGE: prostaglandin E

PGG: principal genetic groups PGL: phenolic glycolipids

PGRS: polymorphic guanine-cytosine rich sequences pI: isoelectric point

pks: polyketide synthase gene

PNB: para-nitrobenzoic acid PPD: purified protein derivative

PPE: proteins that have the motif Pro-Pro-Glu PRA: PCR-restriction enzyme analysis PTFE: polytetrafluoroethylene PZase: pyrazinamidase enzyme

PVNA: polymyxin B, vancomycin, nalidixic acid and amphotericin B rBCG: recombinant BCG

RCF: relative centrifugal force rDNA: ribosomal desoxyribonucleic acid RD: regions of difference

REMA: resazurin microtiter assay

RFLP: restriction fragment length polymorphism RIF: rifampicin

RNA: ribonucleic acid RNAse: ribonuclease

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rrn operon: ribosomal ribonucleic acid operon

RvD: DNA region deleted from H37Rv genome SCG: SNP cluster group

SCID: severe combined immunodeficiency SL: sulfolipid

SLC11A1: solute carrier family 11, member 1 SM: streptomycin

SNP: single nucleotide polymorphism

SpolDB4: fourth international spoligotyping database sSNP: synonymous single nucleotide polymorphism ST: shared-type in SpolDB4

STAT1: signal transducer and activator of transcription 1 TACO: tryptophan aspartate coat protein

TB: tuberculosis

TbD1: M. tuberculosis specific deletion 1 TCH: thiophene-2-carboxylic acid hydrazide TGF-β: transforming growth factor beta

Th1: T helper 1 lymphocyte Th2: T helper 2 lymphocyte TL7H11: thin layer 7H11 agar TLC: thin-layer chromatography TLR: Toll-like receptor

TNF-α: tumor necrosis factor alpha TOF: time of flight

TST: tuberculin skin test US: United States UV: ultraviolet

VDR: vitamin D receptor

VNTR: variable number tandem repeats WHO: World Health Organization XDR: extensively drug resistant

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Content

Chapter 1: History 25

1.1. Primeval tuberculosis 25

1.2. Phthisis/consumption 29

1.3. The White Plague 30

1.4. The discovery of the tubercle bacillus 32

1.5. Sanatorium and initial therapies 34

1.6. 19th_{and 20}th_centuries ₃₈

1.7. A global health emergency 45

References 49

Chapter 2: Molecular Evolution of the Mycobacterium

tuberculosis Complex 53

2.1. A basic evolutionary scheme of mycobacteria 53 2.2. M. tuberculosis complex population molecular genetics 57 2.3. Co-evolution of M. tuberculosis with its hosts 58

2.4. M. tuberculosis through space and time 61

2.5. Looking for robust evolutionary markers 62

2.6. Why repeated sequences were so useful at the beginning 63 2.7. Regions of differences (RDs) and SNPs in M. tuberculosis 63 2.8. Looking for congruence between polymorphic markers 69 2.9. Main lineages within the M. tuberculosis species 72 2.10. When did the bovine-human switch of M. tuberculosis take place? 78 2.11. Comparative genomics and evolution of tubercle bacilli 79 2.12. Short-term evolutionary markers and database building 80

2.13. Conclusion and Perspectives 81

References 83

Chapter 3: The Basics of Clinical Bacteriology 93

3.1. The tubercle bacillus: a continuous taxon 93

3.2. Microscopic morphology 95

3.3. Cell wall structure 97

3.4. Nutritional and environmental requirements for growth 102

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3.6. Metabolic and biochemical markers 106

3.7. Resistance to physical and chemical challenges 107

References 109

Chapter 4: Genomics and Proteomics 113

4.1. Impact of new technologies on Mycobacterium tuberculosis

genomics 113

4.2. M. tuberculosis genome 115

4.3. Gene expression in M. tuberculosis 127

4.4. M. tuberculosis proteome 135

4.5. An insight into M. tuberculosis metabolomics 143

4.6. Concluding remarks 146

References 147

Chapter 5: Immunology, Pathogenesis, Virulence 157

5.1. Immune response against Mycobacterium tuberculosis 157 5.2. Tuberculosis pathogenesis and pathology related to the immune

response 171

5.3. Latency and maintenance of the immune response 183

5.4. Immunotherapy for tuberculosis 184

5.5. Concluding remarks 189

References 189

Chapter 6: Host Genetics and Susceptibility 207

6.1. The difficulty in proving a genetic component for human

susceptibility 207

6.2 Search for mutations and polymorphisms that increase susceptibility 219

6.3. Candidate genes in common tuberculosis 225

6.4 Genes from mouse genetic susceptibility studies 237 6.5. The good, the bad and the maybe, in perspective 244

References 250

Chapter 7: Global Burden of Tuberculosis 263

7.1. Global epidemiology of tuberculosis 263

7.2. Tuberculosis and the interaction with the HIV epidemic 269

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7.4. The new Stop TB strategy 275

References 279

Chapter 8: Tuberculosis caused by Other Members of the M.

tuberculosis Complex 283

8.1. Mycobacterium bovis disease in humans 283

8.2. The BCG vaccine: adverse reactions 290

8.3. Mycobacterium africanum subtypes 293

8.4. Mycobacterium microti disease 295

8.5. Mycobacterium caprae and Mycobacterium pinnipedii 297 8.6. Identification of species within the M. tuberculosis complex 301

References 305

Chapter 9: Molecular Epidemiology: Breakthrough

Achievements and Future Prospects 315

9.1. Introduction 315

9.2. Historical context 317

9.3. Infectiousness of tuberculosis patients 319

9.4. DNA fingerprinting, contact investigation and source case finding 320 9.5. Transmission of drug resistant tuberculosis 323

9.6. Resistance and the Beijing genotype 325

9.7. Genetic heterogeneity of M. tuberculosis and multiple infections 326 9.8. The new standard genetic marker: VNTR typing 329 9.9. DNA fingerprinting to monitor eradication of tuberculosis 331

9.10. Future prospects 332

References 333

Chapter 10: New Vaccines against Tuberculosis 341

10.2. Historical view 342

10.3. Genetic diversity between BCG vaccines 344

10.4. New vaccines: from the bench to clinical trials 345

10.5. Subunit vaccine candidates 348

10.6. Subunit vaccines for boosting BCG 350

10.7. Recombinant BCG vaccines 350

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10.9. Conclusions 354

References 356

Chapter 11: Biosafety and Hospital Control 361

11.1. Biosafety in the hospital 361

11.2. Biosafety in the laboratory 372

References 396

Chapter 12: Conventional Diagnostic Methods 401

12.2. Specimen handling 402

12.3. Smear staining 406

12.4. Adenosine deaminase activity 409

12.5. Culture 410

12.6. Identification 416

References 420

Chapter 13: Immunological Diagnosis 425

13.1. Historical Overview 425

13.2. Current methods of tuberculosis diagnosis 427

13.3. Basis of immunological diagnosis 428

13.4. Serological assays 431

13.5. T cell based assays 433

13.6. Conclusions and Perspectives 437

References 438

Chapter 14: New Diagnostic Methods 441

14.2. Automated culture methods 441

14.3. Nucleic acid amplification methods 450

14.4. Genetic identification methods 461

14.5. Non-conventional phenotypic diagnostic methods 472

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Chapter 15: Tuberculosis in Adults 487

15.2. The initial lesion 487

15.3. The inflammatory response 489

15.4. Tuberculosis infection 490

15.5. Tuberculosis disease 492

15.6. Diagnostic approaches 508

15.7. Treatment of latent tuberculosis infection 516

15.8. Contact tracing and control 518

15.9. The limits between infection and disease 519

References 519

Chapter 16: Tuberculosis in Children 525

16.2. Etiology, transmission and pathogenesis 526

16.3. Primary pulmonary tuberculosis 530

16.4. Non-respiratory disease 533

16.5. Congenital tuberculosis 536

16.6. Diagnosis 537

16.7. Pediatric tuberculosis treatment 544

16.8. Vaccination 552

16.9. Prognosis of pediatric tuberculosis 553

References 554

Chapter 17: Tuberculosis and HIV/AIDS 559

17.1. Epidemiological background 559

17.2. Interactions between M. tuberculosis and HIV infection 560

17.3. Clinical characteristics 561

17.4. Multidrug-resistant tuberculosis and HIV/AIDS 568 17.5. Treatment of tuberculosis in HIV/AIDS patients 574 17.6. Immune reconstitution inflammatory syndrome 579 17.7. Treatment of latent tuberculosis infection in HIV/AIDS patients 580

17.8. Mycobacteriosis in AIDS patients 581

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Chapter 18: Drugs and Drug Interactions 593

18.2. Overview of existing treatment schemes 594

18.3. Drugs: structure, pharmacokinetics and toxicity 601

18.4. Drug resistance mechanisms 612

18.5. Drug interactions 617

18.6. New drugs for tuberculosis 621

18.7. Useful links 627

References 627

Chapter 19: Drug Resistance and Drug Resistance Detection 635

19.2. Drug resistance surveillance 635

19.3. Methods for detection of drug resistance 640

References 655

Chapter 20: New Developments and Perspectives 661

20.1. The scenario 661

20.2. Bacillus and disease under the light of molecular epidemiology 662

20.3. New perspectives in diagnosis 665

20.4. The problem of drug resistance detection 669

20.5. On drug development 670

20.6. On vaccine development 671

20.7. Global management of research & development resources 673

20.8. Useful links 674

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Chapter 1: History

Sylvia Cardoso Leão and Françoise Portaels

Nowhere in these ancient communities of the Eurasian land mass, where it is so common and feared, is there a record of its beginning. Throughout history, it had always been there, a familiar evil, yet forever changing, formless, unknowable. Where other epidemics might last weeks or months, where even the bubonic plague would be marked forever afterwards by the year it reigned, the epidemics of tuberculosis would last whole centuries and even multiples of centuries. Tuberculosis rose slowly, silently, seeping into homes of millions, like an ageless miasma. And once arrived, it never went away again. Year after year, century after century, it tightened its relentless hold, worsening whenever war or famine reduced the peoples' resistance, infecting virtually everybody, inexplicably sparing some while destroying others, bringing the young down onto their sickbeds, where the flesh slowly fell from their bones and they were consumed in the years-long fever, their minds brilliantly alert until, in apocalyptic numbers, they died, like the fallen leaves of a dreadful and premature autumn.

The Forgotten Plague: How the War against Tuberculosis was Won - and Lost Frank Ryan, 1992

Tuberculosis (TB) has a long history. It was present before the beginning of re-corded history and has left its mark on human creativity, music, art, and literature; and has influenced the advance of biomedical sciences and healthcare. Its causative agent, Mycobacterium tuberculosis, may have killed more persons than any other microbial pathogen (Daniel 2006).

1.1. Primeval tuberculosis

It is presumed that the genus Mycobacterium originated more than 150 million years ago (Daniel 2006). An early progenitor of M. tuberculosis was probably contemporaneous and co-evolved with early hominids in East Africa, three million years ago. The modern members of M. tuberculosis complex seem to have origi-nated from a common progenitor about 15,000 - 35,000 years ago (Gutierrez 2005).

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26 History

TB was documented in Egypt, India, and China as early as 5,000, 3,300, and 2,300 years ago, respectively (Daniel 2006). Typical skeletal abnormalities, including Pott’s deformities, were found in Egyptian and Andean mummies (Figure 1-1) and were also depicted in early Egyptian and pre-colombian art (Figure 1-2).

Figure 1-1: Left: Mummy 003, Museo Arqueológico de la Casa del Marqués de San Jorge, Bogota, Colombia. Right: Computerized tomography showing lesions in the vertebral bodies of T10/T11 (reproduced from Sotomayor 2004 with permission).

Figure 1-2: Representation of a woman with pronounced gibbus (Pott´s disease?). Momil culture, 200 BC to 100 AD, Sinú River, Colombia (reproduced from Sotomayor 1992, with permission).

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1.1. Primeval tuberculosis 27 Identification of genetic material from M. tuberculosis in ancient tissues has pro-vided a powerful tool for the investigation of the incidence and spread of human TB in historic periods. It also offers potential new insights into the molecular evo-lution and global distribution of these microbes (see Chapter 2). Research on an-cient DNA poses extreme technical difficulties because of the minute amounts of DNA remains, their oxidation/hydrolysis, and the extremely high risk of contami-nation with modern DNA. For this reason, stringent criteria of authenticity for analysis of ancient DNA were recently proposed, among them: work in physically isolated areas, strict protocols to prevent contamination with modern DNA, the use of negative controls, evaluation of reproducibility in different laboratories, cloning and sequencing, and the study of associated remains (Coper 2002).

Mycobacteria are assumed to be better preserved than other bacteria due to the resistant lipid-rich cell wall and the high proportion of guanine and cytosine in their DNA, which increases its stability. M. tuberculosis are found only in the tissues of an infected host, and the characteristic pathology induced by this strictly mammal-ian pathogen tends to show residual microbial DNA contained in localized lesions. These bacteria are, therefore, ideal microorganisms for studying ancient DNA and were the first to be pursued. These investigations have answered important ques-tions. They proved that TB is an ancient disease with a wide geographical distribu-tion. The disease was widespread in Egypt and Rome (Zink 2003, Donoghue 2004); it existed in America before Columbus (Salo 1994, Konomi 2002, Soto-mayor 2004), and in Borneo before any European contact (Donoghue 2004). The earliest DNA-based documentation of the presence of M. tuberculosis complex organisms was accomplished in a subchondral articular surface from an extinct long-horned Pleistocene bison from Wyoming, US, which was radiocarbon-dated at 17,870 +/- 230 years before the present (Rothschild 2001).

Another important achievement of the studies on ancient DNA was the confirma-tion of the TB diagnosis in human remains that showed the typical pathology. My-cobacterial DNA was detected in bone lesions in the spine of a male human skele-ton from the Iron Age (400-230 BC), found in Dorset, United Kingdom (Taylor 2005); skin samples from the pelvic region of Andean mummies, carbon-dated from 140 to 1,200 AD (Konomi 2002); and calcified pleura from 1,400 year-old remains, found in a Byzantine basilica in the Negev desert (Donoghue 1998). DNA techniques have also shown the presence of mycobacterial DNA, at a lower fre-quency, in bones with no pathological changes, suggesting either dissemination of the TB bacilli immediately prior to death or chronic milliary TB (Zink 2003). Molecular methods other than PCR have also been used to demonstrate the pres-ence of the tubercle bacillus in ancient remains, including mycolic acid analysis by

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28 History

high performance liquid chromatography (HPLC), which is used for authentication of positive PCR findings in calcified pleura remains (Donoghue 1998). Spoligo-typing is a PCR-based technique used for identification and Spoligo-typing of M. tuberculo-sis complex bacteria (see Chapter 9). It is a valuable tool for the study of archeo-logical material, especially when the DNA is highly fragmented, because fragments as small as 55-60 bp long are sufficient to provide a positive result (Donoghue 2004). Spoligotyping was the method used to study the Plesitocene remains of a bison (Rothschild 2001) and was also applied to a subculture of the original tuber-cle bacillus isolated by Robert Koch, confirming its species identification as M. tuberculosis rather than Mycobacterium bovis (Taylor 2003).

Until recently, the search for mycobacterial DNA in human archeological speci-mens failed to find evidence of the presence of M. bovis, a member of the M. tu-berculosis complex with a remarkably wide spectrum of susceptible mammalian hosts, and once considered a putative ancestor of M. tuberculosis (Donoghue 2004). In an up to date publication, the identification of M. bovis DNA in South Siberian human remains was confirmed by amplification of pncA and oxyR genes and analysis of regions of difference (RD) (for a comprehensive review on differ-entiation of species belonging to the M. tuberculosis complex, see Chapters 2 and 8). These findings were obtained from remains that showed skeletal evidence of TB. Carbon-dated from 1,761 to 2,199 years ago, they seem to indicate that this population was continuously exposed to wild or domesticated animals infected with M. bovis, which could have been reservoirs for human infection (Taylor 2007).

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1.2. Phthisis/consumption 29

1.2. Phthisis/consumption

The patients suffer from a latent fever that begins towards evening and vanishes again at the break of day. It is accompanied by violent coughing, which expels thin purulent sputum. The patient speaks with a hoarse voice, breathes with difficulty and has hectically flushed cheeks. The skin on the rest of the body is ashen in color. The eyes have a weary expression, the patient is gaunt in appearance but often displays astonishing physical or mental activity. In many cases, wheezes are to be heard in the chest, and when the disease spreads, sweating is seen on the upper parts of the chest. The patients lose their appetite or suffer hunger pangs. They are often also very thirsty. The ends of the fingers swell and the fingernails curve greatly.

Caelius Aurelianus, 5th century AD (Herzog 1998)

The term phthisis (meaning consumption, to waste away) appeared first in Greek literature. Around 460 BC, Hippocrates identified phthisis as the most widespread disease of the times. It most commonly occurred between 18 and 35 years of age, and was almost always fatal (www.tuberculosistextbook.com/link.php?id=1). He even warned physicians against visiting consumptives in advanced stages of the disease, to preserve their reputation! Although Aristotle (384-322 BC) considered the disease to be contagious, most Greek authors believed it to be hereditary, and a result, at least in part, of the individual's mental and moral weaknesses.

Clarissi-mus Galen (131-201 AD), the most eminent Greek physician after Hippocrates,

defined phthisis as an ulceration of the lungs, chest or throat, accompanied by coughs, low fever, and wasting away of the body because of pus. He also described it as a disease of malnutrition (Pease 1940).

The initial tentative efforts to cure the disease were based on trial and error, and were uniformly ineffective. Heliotherapy was advocated as early as the 5th_century

AD by Caelius Aurelianus. Roman physicians recommended bathing in human urine, eating wolf livers, and drinking elephant blood. In the Middle Ages, it was believed that the touch of the sovereigns of England and France had the power to cure sufferers of the King’s Evil or scrofula (scrophula or struma) - the swellings of the lymph nodes of the neck, frequently related to TB. Depending upon the time and country in which they lived, patients were urged to rest or to exercise, to eat or to abstain from food, to travel to the mountains or to live underground.

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30 History

1.3. The White Plague

Yet the captain of all these men of death that came against him to take him away was consumption, for it was that that brought him down to the grave.

The life and death of Mr. Badman, presented to the world in a familiar dialogue between Mr. Wiseman and Mr. Attentive John Bunyan, 1680

The TB epidemic in Europe, later known as the “Great White Plague”, probably started at the beginning of the 17th_{century and continued for the next 200 years.}

Death from TB was considered inevitable and, by 1650, TB was the leading cause of mortality. The high population density and poor sanitary conditions that charac-terized the enlarging cities of Europe and North America at the time, provided the necessary environment, not met before in world history, for the spread of this air-borne pathogen. The epidemic spread slowly overseas by exploration and coloni-zation.

TB existed in America before Columbus’ arrival but was rare among the natives. The major outbreaks of TB among the native people of North America began in 1880, after they were settled in reservations or forced to live in barracks in prison camps. Death rates increased rapidly, and by 1886, reached 9,000 per 100,000 people (Bates 1993).

TB was also rare among Africans who lived in small remote villages. When ex-posed to the disease by contact with Europeans, these populations experienced a high mortality rate. Africans taken as slaves were free from TB on arrival to the Americas. Then, cases of sub-acute fatal TB developed among them. After their liberation from slavery and movement into the cities, TB morbidity and mortality rose quickly, reaching 700 per 100,000 in 1912 (Bates 1993).

There is also evidence of the presence of the disease in pre-historic Asia, but it was only toward the end of the 19th_{century that peaks in incidence were observed in}

India and China.

In the 18th_{century, TB was sometimes regarded as vampirism. These folk beliefs}

originated from two observations: firstly, following the death from consumption of a family member, household contacts would lose their health slowly. This was attributed to the deeds of the recently deceased consumptive, who returned from the dead as a vampire to drain the life from the surviving relatives. Secondly,

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peo-1.3. The White Plague 31 ple who had TB exhibited symptoms similar to what people considered to be vam-pire traits, such as red, swollen eyes, sensitivity to bright light, pale skin, and a blood-producing cough. They "wasted away" and "lost flesh" and at the same time remained active, and conserved a fierce will to live. This dichotomy of lust and "wasting away" was reflected in the vampires' desire for "food", which forced them to feed off living relatives, who, in turn, suffered a similar wasting away (Sledzik 1994).

Precise pathological and anatomical descriptions of the disease began to appear in the 17th_{century. Franciscus Sylvius de la Böe of Amsterdam (1614-1672) was the}

first to identify the presence of actual tubercles as a consistent and characteristic change in the lungs and other areas of consumptive patients. In his Opera Medica, published in 1679, he also described the progression of the lesions from tubercles to ulcers and cavities. The Latin word tuber means all kinds of degenerative protu-berances or tubercles.

The English physician Richard Morton (1637-1698) confirmed that tubercles were always present in TB of the lungs. He believed that the disease had three stages: inflammation (tubercle formation), ulceration, and phthisis. Both Sylvius de la Böe and Morton regarded the disease as hereditary, although Morton did not rule out transmission by intimate contact.

Gaspard Laurent Bayle (1774-1816) definitely proved that tubercles were not

products, or results, but the very cause of the illness. The name 'tuberculosis' ap-peared in the medical language at that time in connection with Bayle's theory. More precisely, the name 'tuberculosis' was coined in 1839 by the German professor of Medicine Johann Lukas Schönlein (1793-1864), to describe diseases with tuber-cles; but he considered scrofula and phthisis to be separate entities. These ideas were also acknowledged by Giovanne Battista Morgagni in Padua (1682-1771) and Rudolf Virchow in Berlin (1821-1902) (Herzog 1998). In contrast, René

Théophile Hyacinthe Laënnec (1781-1826) from Paris, inventor of the

stetho-scope, and the Viennese Karl von Rokitansky (1804-1878) emphasized the uni-tary nature of both conditions.

The earliest references to the infectious nature of TB appeared in 17th_century

Ital-ian medical literature. An edict issued by the Republic of Lucca in 1699 stated that, "… henceforth, human health should no longer be endangered by objects remain-ing after the death of a consumptive. The names of the deceased should be reported to the authorities and measures undertaken for disinfection" (Herzog 1998).

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32 History

1.4. The discovery of the tubercle bacillus

Not bad air, not just a weakness of the infected human body’s immune system, not any of the myriad theories that had filled the puzzled heads of his audience all of their working lives...but a bacterium. Not just a bacte-rium, but a bacillus the like of which had never been even suspected be-fore, a most singular life form, with a frightening propensity to infect every cat and chicken, pigeon and guinea pig, the white mice and rats, oxen and even two marmosets, into which Koch had injected it.

The Forgotten Plague: How the War against Tuberculosis was Won - and Lost Frank Ryan, 1992

The book De Morbus Contagiosus, written in 1546 by Girolamo Fracastoro (1478-1553), explained the contagious nature of TB. He pointed out that bed sheets and clothing could contain contagious particles that were able to survive for up to two years. The word “particles” may have alluded to chemicals rather than to any kind of living entity.

In his publication A New Theory of Consumptions, in 1720, the English physician

Benjamin Marten (1704-1722) was the first to conjecture that TB could be caused

by “minute living creatures", which, once they had gained entry to the body, could generate the lesions and symptoms of phthisis. He further stated, that consumption may be caught by a sound person by lying in the same bed, eating and drinking or by talking together so close to each other as to “draw in part of the breath a con-sumptive patient emits from the lungs”.

In 1865, the French military doctor Jean-Antoine Villemin (1827-1892) demon-strated that consumption could be passed from humans to cattle, and from cattle to rabbits. On the basis of this revolutionary evidence, he postulated that a specific microorganism caused the disease. At this time William Budd (1811-1880) also concluded from his epidemiological studies that TB was spread through society by specific germs.

On the evening of March 24, 1882, in Berlin, before a skeptical audience composed of Germany's most prominent men of science from the Physiological Society,

Rob-ert Koch (1843-1910) (www.tuberculosistextbook.com/link.php?id=2) made his famous presentation Die Aetiologie der Tuberculose. Using solid media made of potato and agar, Koch invented new methods of obtaining pure cultures of bacteria. His colleague Julius Richard Petri (1852-1921) developed special flat dishes (Petri dishes), which are still in common use, to keep the cultures. Koch also

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de-1.4. The discovery of the tubercle bacillus 33 veloped new methods for staining bacteria, based on methylene blue, a dye devel-oped by Paul Ehrlich (1854-1915) (www.tuberculosistextbook.com/link.php?id=3), and counterstained with vesuvin. "Under the microscope the structures of the animal tissues, such as the nucleus and its breakdown products are brown, while the tubercle bacteria are a beautiful blue", he wrote in the paper that followed his dramatic presentation that March evening (Koch 1882).

He had brought his entire laboratory with him: his microscopes, test tubes, small flasks with cultures, and slides of human and animal tissues preserved in alcohol. Showing the presence of the bacillus was not enough. He wanted his audience to note that bacteria were always present in TB infections and could be grown on solidified serum slants, first appearing to the naked eye in the second week. Then, he showed that, by inoculating guinea pigs with tuberculous material obtained from lungs, intestines, scrofula or brains of people and cattle that have died from TB, the disease that developed was the same, and cultures obtained from the experimental animals were identical on the serum slopes. Koch continued his speech, proving that whatever the dose and/or route he used, no matter what animal species he in-oculated, the results were always the same. The animals subsequently developed the typical features of TB. He concluded saying that “…the bacilli present in tu-berculous lesions do not only accompany tuberculosis, but rather cause it. These bacilli are the true agents of tuberculosis” (Kaufmann 2005).

Koch fulfilled the major prerequisites for defining a contagious disease that had, in fact, been proposed by his former mentor Jacob Henle (1809-1885). The re-knowned Koch's postulates (or Henle-Koch postulates) were then formulated by Robert Koch and Friedrich Loeffler (1852-1915) in 1884, and finally polished and published by Koch in 1890. The postulates consist of four criteria designed to es-tablish a causal relationship between a causative microbe and a disease:

• The organism must be found in all animals suffering from the disease, but not in healthy animals

• The organism must be isolated from a diseased animal and grown in pure culture

• The cultured organism should cause disease when introduced into a healthy animal

• The organism must be re-isolated from the experimentally infected animal. In 1890, at the 10th_{International Congress of Medicine held in Berlin, Koch}

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34 History

when given both pre- and post-exposure. It was called 'tuberculin' and was prepared from glycerol extracts of liquid cultures of tubercle bacilli. Clinical trials using tuberculin as a therapeutic vaccine were soon initiated. The results were published in 1891 and revealed that only few persons were cured, at a rate not different from that of untreated patients. But, although results for treatment were disappointing, tuberculin was proven valuable for the diagnosis of TB (Kaufmann 2005).

One of Koch’s papers (Koch 1891), describing the preparation and partial purifica-tion of tuberculin served as the first descrippurifica-tion of the producpurifica-tionof the partially purified derivative (PPD) of tuberculin, presently used in the Mantoux test, also known as the Tuberculin Skin Test, Pirquet test, or PPD test (see Chapter 13).

1.5. Sanatorium and initial therapies

…not for nothing was it famous far and wide. It had great properties. It accelerated oxidization, yet at the same time one put on flesh. It was capa-ble of healing certain diseases which were latent in every human being, though its first effects were strongly favorable to these, and by dint of a general organic compulsion, upwards and outwards, made them come to the surface, brought them, as it were, to a triumphant outburst.

- Beg pardon -- triumphant?

- Yes; had he never felt that an outbreak of disease had something jolly about it, an outburst of physical gratification?

The Magic Mountain [Der Zauberberg] Thomas Mann, 1924 Translated from the German by H. T. Lowe-Porter, 1953 Dialogue between Hans Castorp and consul Tienappel

The introduction of the sanatorium cure provided the first widely practiced ap-proach to anti-tuberculosis treatment. Hermann Brehmer (1826-1889) a Silesian botany student suffering from TB, was instructed by his doctor to seek out a healthier climate. He traveled to the Himalayas where he studied the mountain’s flora. He returned home cured and began to study medicine. In 1854, he presented his medical dissertation Tuberculosis is a Curable Disease. Brehmer then opened an in-patient hospital in Gorbersdorf, where patients received good nutrition and

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1.5. Sanatorium and initial therapies 35 were continuously exposed to fresh air. This became the model for all subsequent sanatoria, including the one depicted in Thomas Mann’s The Magic Mountain. A young doctor named Edward Livingston Trudeau (1848-1915) established the most famous sanatorium in the United States at Saranac Lake, in New York's Adi-rondak Mountains (http://www.trudeauinstitute.org/info/history/history.htm). He also suffered from TB and, in 1882, became aware of Koch's experiments with TB bacteria and of Brehmer's sanatorium. Trudeau established the Saranac Laboratory for the Study of Tuberculosis. It was the first institution devoted to TB research in the United States (US).

Sanatoria, increasingly found at that time throughout Europe and the US, provided a dual function. Firstly, they protected the general population by isolating the sick persons, who were the source of infection. Secondly, they offered TB patients bed-rest, exercise, fresh-air, and good nutrition, all of which assisted the healing proc-ess. Many of them improved and returned to "life in the flatland"; many did not. The TB specialist, the phthisiologist, was responsible for the complete physical and mental care of the patient and the separation of TB care from the practicing clini-cian became commonplace.

Architectural features were essential to early sanatorium design (Figure 1-3). These included deep verandas, balconies, covered corridors, and garden shelters, fur-nished with reclining couches for the “Cure”, the obligatory two-hour period of rest in the open air that was frequently observed in silence (Figure 1-4). Furniture for TB patients had to be robust, able to be thoroughly cleaned and disinfected, and shaped with a concern for the patient’s anthropometric needs.

Alvar Aalto (1898-1976), Jan Duiker (1890-1935) and Charles-Edouard Jean-neret (Le Corbusier) (1887-1965) were modernist architects and designers that

adapted and interpreted the ideas of functionality and rationality derived from con-cepts used in the treatment of TB, and their designs for buildings and furniture became icons of modernism. Aalto won the competition of Architecture, Interior Design and Furniture Design for the constuction ofthe Paimio Tuberculosis Sana-torium in 1928, and Duiker designed the Zonnestraal SanaSana-torium. The symbolic association of light and air with healing made a profound influence on modernist ideas for design. Flat roofs, balconies, terraces and reclining chairs were subse-quently adopted for the design of fashionable buildings in rapidly expanding cities such as Paris and Berlin (Campbell 2005).

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36 History

Figure 1-3: Sanatorio Pineta del Carso, Trieste, Italy.

Figure 1-4: Sanatorio Pineta del Carso. Bed-rest, fresh air and good nutrition were the hall-marks of sanatorium cure.

Probably, it will never be known whether sanatorium treatment was a success or a failure, because no study was undertaken comparing the rates of mortality of sana-torium patients with those of TB patients who were similar in age, sex, and eco-nomic position, but who remained untreated or were treated by other methods.

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1.5. Sanatorium and initial therapies 37 Nevertheless, physicians with a long and intimate experience with the disease were unanimous in the opinion that open-air treatment was an improvement for the aver-age consumptive (McCarthy 2001).

During the early ’60s, many sanatoria started to close. By the middle of that decade only a few beds remained available for patients suffering from TB. Yet, the real end of the TB sanatorium began even earlier, when the depressing era of helpless-ness in the face of advanced TB was substituted by active therapy.

The Italian physician Carlo Forlanini (1847-1918) discovered that the collapse of the affected lung tended to have a favorable impact on the outcome of the disease. He proposed to reduce the lung volume by artificial pneumothorax and surgery, methods that were applied worldwide after 1913. These and other initial therapies are now considered dangerous and, at least, controversial:

• Artificial pneumothorax - pleural cavities were filled with gas or filtered air, with the result of splinting and collapsing that lung (Sharpe 1931). • Bilateral pneumothorax - only parts of the lungs were collapsed in such a

way that the patient could still live a relatively normal live. The patient suf-fered from shortness of breath caused by the reduction in the gas exchange surface.

• Thoracoplasty - ribs from one side of the thorax were removed in order to collapse the infected portion of the lung permanently (Samson 1950). • Gold Therapy - Holger Mollgaard (1885-1973) from Copenhagen

intro-duced the compound sanocrysin in 1925, which is a double thiosulphate of gold and sodium. He tested the compound on animals and considered it safe for human use. However, it was too toxic even in low doses. A con-trolled trial, completed in the US in 1934, proved the toxic effects of gold therapy. Within a year, most European countries had ceased to use it (Be-denek 2004).

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38 History

1.6. 19

th

and 20

th

centuries

There is a dread disease which so prepares its victim, as it were, for death; which so refines it of its grosser aspect, and throws around familiar looks unearthly indications of the coming change; a dread disease, in which the struggle between soul and body is so gradual, quiet, and solemn, and the result so sure, that day by day, and grain by grain, the mortal part wastes and withers away, so that the spirit grows light and sanguine with its lightening load, and, feeling immortality at hand, deems it but a new term of mortal life; a disease in which death and life are so strangely blended, that death takes the glow and hue of life, and life the gaunt and grisly form of death; a disease which medicine never cured, wealth never warded off, or poverty could boast exemption from; which sometimes moves in giant strides, and sometimes at a tardy sluggish pace, but, slow or quick, is ever sure and certain.

Nicholas Nickleby Charles Dickens, 1870

When, in 1820, the poet John Keats (1795-1821) coughed a spot of bright red blood, he told a friend, "It is arterial blood. I cannot be deceived. That drop of blood is my death warrant. I must die". He died within a year, at just 25 years of age. Keats never wrote specifically about phthisis, but his life and his works be-came a metaphor that helped transform the physical disease "phthisis” into its spiritual offspring, "consumption".

The central metaphor of consumption in the 19th_{century was the idea that the}

phthisic body is consumed from within by its passions. Spes phthisica (spes - hope + phthisis - consumption) was a condition believed to be peculiar to consumptives in which physical wasting led to a sense of well-being and happiness, an euphoric blossoming of passionate and creative aspects of the soul. While the body expired from phthisis, the prosaic human became poetic and the creative soul could be released from the fevered combustion of the body. The paleness and wasting, the haunted appearance, the burning sunken eyes, the perspiring skin - all hallmarks of the disease - came to represent feminine beauty, romantic passion, and fevered sexuality (Morens 2002).

In the 19th_{century, it seemed as if everyone was slowly dying of consumption. The}

disease became to be viewed in popular terms, first as romantic redemption (Figure 1-5), then as a reflection of societal ills (Figure 1-6) (Morens 2002). In Alexandre

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1.6. 19th and 20th centuries 39 by love and made unforgettable by progressive consumption. It was adapted to the theatre and the movies and also inspired Giuseppe Verdi’s opera “La Traviata”. The plot develops around the consequences of the heroine’s scandalous past, which prevents her marriage to an honorable youngster whose father objects to the rela-tionship. Redemption is possible only through death, and, in taking her life, con-sumption also serves as a vehicle for punishment.

Figure 1-5: Romantic view of TB: “The Lady of the Camellias” represented by Brazilian actress Cacilda Becker under Italian director Luciano Salce, in São Paulo, Brazil (1952).

By 1896, the cause of consumption had been discovered, and TB was definitively linked to poverty and industrial disfigurement, child labor, and sweatshops. A con-tagious disease and shameful indicator of class, it was no longer easily romanti-cized in conventional artistic terms. Giacomo Puccini’s “La bohème” (1896) por-trays TB in a new environment, affecting street artists struggling with poverty and disease (Figure 1-6).

At the end of the 19th_{century, the association of TB with poor living conditions and}

hygiene brought to life the differentiation and societal repulsion of diseased per-sons, considered to be responsible for a social wickedness. Unlike the previous image (sick people as victims), they began to be viewed as dangerous, because they were capable of spreading the disease to those who did not share their living condi-tions. TB was changed from a social disease to an individual one and the patient was at the same time offender and victim of this social ailment.

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40 History

A list of famous people and celebrities who had, or are believed to have had TB is available on Wikipedia at www.tuberculosistextbook.com/link.php?id=4 and at

www.tuberculosistextbook.com/link.php?id=5

Figure 1-6: Social aspect of TB: Second act of “La bohéme”, showing Quartier Latin, with a great crowd on the street and sellers praising their wares.

After the establishment, in the ’80s, that the disease was contagious, TB was made a notifiable disease. A further significant advance came in 1895, when Wilhelm

Konrad von Röntgen (1845-1923) discovered X-rays

(www.tuberculosistextbook.com/link.php?id=6). After this, the progress and sever-ity of a patient's disease could be accurately documented and reviewed.

At the beginning of the 20th_{century, public health authorities realized that TB was}

preventable and that it was not directly inherited. Several associations were set up to educate the community at large. Books educated people about bad food, bad air and unhealthy drinking water. Public health reformers used illustrative posters and stamps (see http://www.nlm.nih.gov/exhibition/visualculture/tuberculosis.html) as a means of communication, advertisement, and persuasion. This new medium quickly became an effective educational and fundraising tool in the widespread campaign against TB.

Centralized official and/or non-governmental agencies for coordination and com-munication were organized and called for conferences specifically focused on TB. At the Central Bureau for the Prevention of Tuberculosis, which was formalized in Berlin in 1902, Dr. Gilbert Sersiron suggested that, as the fight against TB was a crusade, it would be appropriate to adopt the emblem of a crusader, the Duke of

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1.6. 19th and 20th centuries 41 Lorraine. Godfrey of Bouillon (1060-1100), Duke of Lorraine, was the first Chris-tian ruler of Jerusalem and his banners bearing the double-barred cross signified courage and success to crusaders. Dr. Sersiron's recommendation was adopted and the double-barred cross became the worldwide symbol of the fight against TB (Figure 1-7).

Figure 1-7: double-barred cross, symbol of anti-tuberculosis crusade

Periodic international conferences systematically addressing clinical, research and sociological aspects of TB were held until the outbreak of World War I in 1914. After the war, in 1920, a conference on TB was held in Paris with participation of delegates from 31 countries, among them Australia, Bolivia, Brazil, Chile, China, Colombia, Cuba, Guatemala, Japan, Panama, Paraguay, Iran and Thailand, in addi-tion to those of Europe and North America, thus establishing the Internaaddi-tional Un-ion Against Tuberculosis and Lung Disease (IUATLD,

http://www.iuatld.org/index_en.phtml) in its present form.

With Edward Jenner’s (1749-1823) successful invention, showing that infection with cowpox would give immunity against smallpox in humans, many doctors placed their hopes on the use of M. bovis – the agent that causes bovine TB – for the development of a vaccine against human TB. However M. bovis was equally contagious in humans. From 1908 until 1919, Albert Calmette (1863-1933) (http://www.pasteur.fr/infosci/archives/cal0.html) and Camille Guérin (1872-1961) (http://www.pasteur.fr/infosci/archives/gue0.html) in France serially passed a pathogenic strain of M. bovis 230 times, resulting in an attenuated strain called Bacille Calmette-Guérin or BCG, which was avirulent in cattle, horses, rabbits, and

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42 History

guinea pigs. BCG was first administered to humans in 1921 and it is still widely applied today (see Chapter 10).

Then, in the middle of World War II, came the final breakthrough, the greatest challenge to the bacterium that had threatened humanity for thousands of years -chemotherapy. In 1943, streptomycin, a compound with antibiotic activity, was purified from Streptomyces griseus by Selman A. Waksman (1888-1973) (www.tuberculosistextbook.com/link.php?id=7) and his graduate student Albert

Shatz (1920-2005) (Shatz 1944a). The drug was active against the tubercle bacillus

in vitro (Schatz 1944b) and following infection of guinea pigs (Feldman 1944). It was administered to a human patient at the end of 1944 (Hinshaw 1944). Two pio-neering clinical studies were conducted on the treatment of TB patients with strep-tomycin, one in Europe and the other in the US (Medical Research Council 1948, Pfuetze 1955). A considerable improvement in the disease was observed in patients on streptomycin therapy, but after the first months, some patients began to deterio-rate and these pioneering studies properly interpreted such treatment failure as a consequence of development of resistance to the drug.

In 1943, Jörgen Lehmann (1898-1989) wrote a letter to the managers of a phar-maceutical company, Ferrosan, suggesting the manufacture of the para-amino salt of aspirin because it would have anti-tuberculous properties (Ryan 1992). The Swedish chemist based his theory on published information, stressing the avidity of tubercle bacilli to metabolize salicylic acid. He realized that by changing the structure of aspirin very slightly, the new molecule would be taken up by the bacte-ria in just the same way, but would not work like aspirin and would rather block bacterial respiration. Para-aminosalicylic acid (PAS) was produced and first tested as an oral therapy at the end of 1944. The first patient treated with PAS made a dramatic recovery (Lehmann 1964). The drug proved better than streptomycin, which had nerve toxicity and to which M. tuberculosis could easily develop resis-tance.

In the late ’40s, it was demonstrated that combined treatment with streptomycin and PAS was superior to either drug alone (Daniels 1952). Yet, even with the com-bination of the two drugs, TB was not defeated. Overall, about 80 % of sufferers from pulmonary TB showed elimination of their germs; but 20 % were not cured, especially those with extensive disease and cavitation (Ryan 1992).

Two further findings were very important for TB treatment. Firstly, between 1944 and 1948, the action of nicotinamide on the TB bacillus was discovered by two different groups, but this discovery was not widely appreciated at the time. Sec-ondly, in 1949, reports stated that the Germans had treated some 7,000 tuberculous